The development of small molecule chemical probes or therapeutics that target RNA remains a significant challenge despite the great interest in such compounds. The most significant barrier to compound development is a lack of knowledge of the chemical and RNA motif spaces that interact specifically.
RNA plays diverse and important roles in biological processes (1). Aberrant RNA function causes many severe diseases (2). For example, microRNA disregulation can contribute to cancer (3) and single nucleotide mutations in mRNAs cause beta-thalassemia and inherited breast cancer (4). RNA trinucleotide repeat expansions (termed r(NNN)exp where each rN signifies a ribonucleotide of the repeated sequence) cause various neurological disorders (5) including Fragile X Syndrome (FXS), Fragile X-associated Tremor Ataxia Syndrome (FXTAS), myotonic dystrophy type 1 (DM1), and Huntington's disease (HD).
Although RNA transcripts with expanded repeats cause the diseases mentioned above, the physiological response to the repeats and thus the causes of disease are quite different. Differences are mainly due to the location of the expanded repeats in a given mRNA. For example, HD is caused by an expansion of r(CAG) in the coding region of huntingtin mRNA. In the most well established mechanism of HD, disease is caused when expanded r(CAG) repeats are translated into a toxic polyQ version of huntingtin (6). Thus, HD is caused by a gain-of-function at the protein level. In FXS, >200 copies of r(CGG) in the 5′ untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) mRNA causes disease by recruiting the ‘RNA-induced initiator of transcriptional gene silencing’ (RITS) complex. The RITS complex then recruits DNA methyltransferase(s) (DMTases) and/or histone methyltransferases (HMT) to initiate local methylation of the FMR1 gene, causing transcriptional silencing (7). Thus, FXS is caused by a loss-of-function. Lastly, FXTAS and DM1 are caused when expanded repeats present in UTR's sequester proteins that are involved in pre-mRNA splicing regulation (8, 9). Sequestration of these proteins causes the aberrant splicing of a variety of pre-mRNAs, leading to the expression of defective proteins. Thus, FXTAS and DM1 are caused by an RNA gain-of-function.
FXTAS is a late onset (over age 50) neurological condition that affects balance, tremor, and memory. It affects 1 in 3000 men and 1 in 5000 women. FXTAS is caused by expanded CGG-repeat (55-200) alleles in the 5′ untranslated region (UTR) of the fragile X mental retardation 1 (FMR1) gene located on the X chromosome. Gain-of-function of r(CGG)exp is a general pathogenic mechanism of FXTAS similar to myotonic dystrophy. Evidence for RNA gain-of-function comes from animal models and cell-based assays. For example, insertion of untranslated r(CGG)exp of the length that cause FXTAS into mice and Drosophila cause deleterious effects like those observed in humans that have FXTAS. In cell-based models, r(CGG)exp form nuclear inclusions, and the size of inclusions scales with the length of the repeat and the age of death from the disease.
A more detailed mechanism for the RNA gain-of-function has recently been elucidated from studies of patient-derived tissues and model cell lines. In studies by the Charlet group, it was shown that r(CGG)exp first recruits DGCR8, followed by recruitment of the Src-Associated substrate during mitosis of 68 kDa (Sam68) protein. The RNA-protein complex is a scaffold for the assembly of other proteins such as muscleblind-like 1 protein (MBNL1) and heterogeneous nuclear ribonucleoprotein (hnRNP). Sam68 is a nuclear RNA-binding protein involved in alternative splicing regulation, and the sequestration of Sam68, MBNL1, and hnRNP by r(CGG)exp leads to the pre-mRNA splicing defects observed in FXTAS patients (see FIG. 1). Targeting r(CGG)exp to inhibit DGCR8 and Sam68 binding is an attractive treatment for FXTAS.
Despite the contribution of expanded RNA repeats to diseases, there are few compounds that target these RNAs in particular and non-ribosomal RNAs in general. Our group recently reported two approaches to design of small molecules (10, 11) and modularly assembled compounds (12) that bind RNA and modulate its function in vivo. In particular, we have used information about RNA motif-small molecule interactions (13-15) and chemical similarity searching (16-19) to design bioactive ligands that target r(CUG)exp and r(CAG)exp, which cause DM1 and HD, respectively (10-12).